Two overarching themes of the biomedical research of this Superfund Program addressed in this project relate to a) the metabolism of arsenic (As) and b) As-induced oxidative stress. There is significant variability in progression from As exposure to clinical manifestations of disease. Several studies have led to the hypothesis that nutritional status may account for a substantial portion of this variability. Inorganic As is methylated via one-carbon metabolism, a biochemical pathway that is dependent on folate for recruitment of one-carbon groups. We wish to expand our studies, which have begun to characterize the impact of nutritional regulation of one-carbon metabolism on the inter-individual variability in As methylation. Glutathione (GSH), a key component of the primary antioxidant defense mechanism, and the electron donor for As reduction, is synthesized from homocysteine, and this synthesis is regulated by intermediates of onecarbon metabolism. A great deal of basic research, including salient work from members of our group, points to the growing belief that As depletes glutathione (GSH) and induces oxidative stress. However, the relationship between As exposure and oxidative stress has not been rigorously examined in human populations. The first specific aim of this proposal will utilize the repository of biological samples established by the Cohort Study (Project #2) to conduct a nested case-control study to identify modifiable risk factors (e.g. oxidative stress and/or hyperhomocysteinemia) related to increased susceptibility to As-induced skin lesions. The remaining specific aims will take advantage of the expansion of our study area (and installation of Asfree tube wells) in Projects #3 and #7 to recruit 375 new adults who are currently exposed to As. In Specific Aim 2, we will address a fundamental question: To what extent do urinary As metabolites reflect As metabolites in the circulation? In Specific Aim 3, we will conduct a cross-sectional study to test the hypotheses that higher concentrations of s-adenosylhomocysteine (SAH) and lower concentrations of GSH are associated with reduced As methylation. In Specific Aim 4, we propose to examine dose-response relationships between As exposure and oxidative stress. Finally, we will test the hypothesis that reduction of As exposure alleviates oxidative stress. The proposed studies have the potential to a) substantiate that As induces oxidative stress and depletes GSH in a human population, b) link As-induced oxidative stress and/or nutritional status to an arsenic-related clinical outcome, and c) expand our understanding of the mechanisms underlying these processes. Such findings would have significant implications for the identification of potential targeted interventions for preventing As-toxicity.